通脉养心丸对ApoE-/-小鼠动脉斑块形成的影响及机制研究

Study of the effect and related mechanism of Tongmai Yangxin Pill on atherosclerosis in ApoE-/- mice

关键词:  动脉粥样硬化  通脉养心丸  基因芯片  ApoE^-/-小鼠

Key words:  atherosclerosis  Tongmai Yangxin Pill  microarray  ApoE^-/- mice

摘要:

[目的] 观察通脉养心丸对载脂蛋白E基因敲除（ApoE^-/-）小鼠动脉斑块形成的影响并对其可能的作用机制进行探索。[方法] 将ApoE^-/-小鼠分为4组，分别为模型组、通脉养心丸2.0 g/kg组；通脉养心丸1.0 g/kg组、通脉养心丸0.5g/kg组，C57BL/6J小鼠为空白对照组。高脂饲养ApoE^-/-小鼠，复制动脉粥样硬化（AS）模型。末次给药1 h后，生化法测定血清中总胆固醇（TC）及三酰甘油（TG）水平；分别采用油红O染色法及苏木精-伊红（HE）染色法，观察斑块形成及动脉缩窄程度；采用Affymetrix Mouse Genome对小鼠动脉组织基因谱进行检测，进行差异基因分析、聚类分析、Ingenuity Canonical通路分析，探究通脉养心丸药效作用机制。并采用实时定量聚合酶链式反应（qRT-PCR）和酶联免疫吸附剂测定（ELISA）法对关键蛋白或基因进行验证。[结果] 与空白对照组比较，模型组小鼠血清TC、TG水平显著升高，动脉大面积斑块形成，动脉明显缩窄，通脉养心丸能有效改善上述情况；差异基因富集分析表明，通脉养心丸可能通过影响PPARα/RXRα、基质金属蛋白酶信号通路、血小板源性生长因子信号通路等产生抑制斑块形成作用；通脉养心丸能使过氧化物酶体增殖物激活受体α（PPARα）基因表达显著上调，并使血浆中p-核因子κB（p-NF-κB）、肿瘤坏死因子α（TNF-α）、白介素6（IL-6）、白介素1β（IL-1β）水平显著降低。[结论] 通脉养心丸能够有效抑制动脉斑块形成，活化PPARα，抑制炎症因子表达是药效产生的确切途径之一。

Abstract:

[Objective] To observe the effect of Tongmai Yangxin Pill treated the arterial plaque in ApoE^-/- mice and to explore the mechanism.[Methods] The ApoE^-/- mice were randomly divided into four groups,including model group,Tongmai Yangxin Pill 2.0 g/kg group,Tongmai Yangxin Pill 1.0 g/kg group,Tongmai Yangxin Pill 0.5 g/kg group. C57BL/6J mice were control group. ApoE^-/- mice were fed with high fat diet to establish atherosclerosis model. One hour after the last administration,the levels of total cholesterol (TC) and triglycerides (TG) in serum were determined by biochemical method. Oil red O staining and HE staining were used to observe the plaque formation and the degree of arterial constriction. Affymetrix mouse genome was used to detect the gene spectrum of mouse artery tissue,and the differential gene analysis,cluster analysis and ingenuity canonical pathway analysis were carried out to explore the pharmacodynamic mechanism of Tongmai Yangxin Pill. RT-PCR and ELISA were used to verify the key proteins or genes.[Results] Compared with the control group,Tongmai Yangxin Pill could effectively improve the formation of large area plaque and significantly improve the narrow artery in the model group. The differential gene enrichment analysis showed that Tongmai Yangxin Pill could inhibit the formation of plaque by affecting PPAR α/RXR α,matrix metalloproteinase signaling pathway and platelet-derived growth factor signaling pathway. The expression of PPAR α gene was significantly up-regulated and the levels of p-NF-κB,TNF-α,IL-6 and IL-1 β were significantly decreased.[Conclusion] Tongmai Yangxin Pill can effectively inhibit the formation of arterial plaque,activate PPAR α and inhibit the expression of inflammatory factors,which is one of the exact ways of drug effect.